DETERMINATION OF HEPATITIS-C VIRUS GENOTYPES IN THE UNITED-STATES BY CLEAVASE FRAGMENT LENGTH POLYMORPHISM ANALYSIS

We describe the application of a new DNA-scanning method, which has be en termed Cleavase Fragment Length Polymorphism (CFLP; Third Wave Tech nologies, Inc., Madison, Wis.), for the determination of the genotype of hepatitis C virus (HCT). CFLP analysis results in the generation of structural fingerprints that allow discrimination of different DNA se quences. We analyzed 251-bp cDNA products generated by reverse transcr iption-PCR of the well-conserved 5'-noncoding region of HCV. We determ ined the genotypes of 87 samples by DNA sequencing and found isolates representing 98% of the types typically encountered in the United Stat es, i.e., types 1a, 1b, 2a/c, 2b, 3a, and 4. Blinded CFLP analysis of these samples was 100% concordant with DNA sequencing results, such th at closely related genotypes yielded patterns with strong familial res emblance whereas more divergent sequences yielded patterns with pronou nced dissimilarities. In each case, the aggregate pattern,fas indicati ve of genotypic grouping, while finer changes suggested subgenotypic d ifferences. We also assessed the reproducibility of CFLP analysis in H CV genotyping by analyzing three distinct isolates belonging to a sing le subtype. These three isolates yielded indistinguishable CFLP patter ns, as did replicate analysis of a single isolate. This study demonstr ates the suitability of this technology for HCV genotyping and suggest s that it may provide a low-cost, high-throughput alternative to DNA s equencing or other, more costly or cumbersome genotyping approaches.